Omega-hydroxylase-related fusion polypeptide variants with improved properties

Abstract

The disclosure relates to omega-hydroxylase-related fusion polypeptides that result in improved omega-hydroxylated fatty acid derivative production when expressed in recombinant host cells. The disclosure further relates to microorganisms for expressing the omega-hydroxylase-related fusion polypeptides for the production of omega-hydroxylated fatty acid derivatives.

What is claimed is: 
     
       1. A cytochrome P450 CYP153A reductase (CYP153A-reductase) hybrid fusion polypeptide variant having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 38 and having at least one mutation at an amino acid position corresponding to amino acid position: 9, 10, 11, 12, 13, 14, 28, 61, 77, 119, 159, 231, 233, 327, 413, 703, 745, 747, 749, 757, 770, 771 or 784, wherein the CYP153A-reductase hybrid fusion polypeptide variant has a mutation at each of amino acid positions:
 (a) 12, 27, 28, 119, 141, 157, 159, 231, 233, and 244 of SEQ ID NO: 38; 
 (b) 12, 28, 119, 140, 157, 159, 233, 244, 254, and 407 of SEQ ID NO: 38; 
 (c) 12, 27, 111, 119, 141, 157, 159, 231, 233, 244, and 254 of SEQ ID NO: 38; 
 (d) 12, 28, 119, 140, 149, 157, 159, 231, 233, and 407 of SEQ ID NO: 38; 
 (e) 12, 27, 28, 119, 140, 157, 159, 233, 244, and 407 of SEQ ID NO: 38; 
 (f) 10, 11, 12, 28, 119, 141, 159, 231, 233, 244, and 407 of SEQ ID NO: 38; 
 (g) 11, 12, 27, 28, 119, 141, 157, 159, 197, 231, 233, 244, 407, and 477 of SE ID NO: 38; 
 (h) 11, 12, 28, 119, 141, 157, 159, 197, 231, 233, 244, and 407 of SEQ ID NO: 38; or 
 (i) 11, 12, 27, 28, 119, 141, 149, 157, 159, 231, 233, and 407 of SEQ ID NO: 38; 
 wherein the CYP153A-reductase hybrid fusion polypeptide variant catalyzes the conversion of a fatty acid to an omega-hydroxylated fatty acid. 
 
     
     
       2. The CYP153A-reductase hybrid fusion polypeptide variant of  claim 1 , wherein:
 (a) the mutations at amino acid positions 12, 27, 28, 119, 141, 157, 159, 231, 233, and 244 of SEQ ID NO: 38 are Q12W, R27L, Q28M, K119R, V141T, S157R, V159M, A231Y, S233L, and A244R, respectively; 
 (b) the mutations at amino acid positions 12, 28, 119, 140, 157, 159, 233, 244, 254, and 407 of SEQ ID NO: 38 are Q12W, Q28M, K119R, S140N, S157R, V159M, S233L, A244R, R254G, and N407G, respectively; 
 (c) the mutations at amino acid positions 12, 27, 111, 119, 141, 157, 159, 231, 233, 244, and 254 of SEQ ID NO: 38 are Q12W, R27L, F111A, K119R, V141T, S157R, V159M, A231Y, S233L, A244R, and R254G, respectively; 
 (d) the mutations at amino acid positions 12, 28, 119, 140, 149, 157, 159, 231, 233, and 407 of SEQ ID NO: 38 are Q12W, Q28M, K119R, S140N, P149G, S157R, V159M, A231Y, S233L, and N407G, respectively; 
 (e) the mutations at amino acid positions 12, 27, 28, 119, 140, 157, 159, 233, 244, and 407 of SEQ ID NO: 38 are Q12W, R27L, Q28M, K119R, S140N, S157R, V159M, S233L, A244R, and N407G, respectively; 
 (f) the mutations at amino acid positions 10, 11, 12, 28, 119, 141, 159, 231, 233, 244, and 407 of SEQ ID NO: 38 are D10Y, I11L, Q12W, Q28M, K119R, V141T, V159M, A231Y, S233L, A244R, and N407G, respectively; 
 (g) the mutations at amino acid positions 11, 12, 27, 28, 119, 141, 157, 159, 197, 231, 233, 244, 407, and 477 of SEQ ID NO: 38 are I11L, Q12W, R27L, Q28M, K119R, V141T, S157R, V159M, A197T, A231Y, S233L, A244R, N407G, and P477G, respectively; 
 (h) the mutations at amino acid positions 11, 12, 28, 119, 141, 157, 159, 197, 231, 233, 244, and 407 of SEQ ID NO: 38 are I11L, Q12W, Q28M, K119R, V141T, S157R, V159M, A197T, A231Y, S233L, A244R, and N407G, respectively; and 
 (i) the mutations at amino acid positions 11, 12, 27, 28, 119, 141, 149, 157, 159, 231, 233, and 407 of SEQ ID NO: 38 are I11L, Q12W, R27L, Q28M, K119R, V141T, P149G, S157R, V159M, A231Y, S233L, and N407G, respectively. 
 
     
     
       3. The CYP153A-reductase hybrid fusion polypeptide variant of  claim 1 , wherein the CYP153A-reductase hybrid fusion polypeptide variant comprises a mutation at amino acid position 12 and at each of amino acid positions:
 (a) 27, 119, 140, 157, 159, 233, and 244 of SEQ ID NO: 38; 
 (b) 28, 61, 119, 231, 309, 413, and 480 of SEQ ID NO: 38; 
 (c) 119, 231, and 480 of SEQ ID NO: 38; 
 (d) 28, 77, 119, 141, 231 and 407 of SEQ ID NO: 38; 
 (e) 28, 61, 141, 231, and 407 of SEQ ID NO: 38; 
 (f) 28, 119, 231, and 244 of SEQ ID NO: 38; 
 (g) 28, 407, and 480 of SEQ ID NO: 38; 
 (h) 141, 231, 413, and 481 of SEQ ID NO: 38; 
 (i) 28, 111, 231, and 407 of SEQ ID NO: 38; 
 (j) 28, 61, 140, and 149 of SEQ ID NO: 38; 
 (k) 28, 77, 119, 159, 231, 254, 407, and 480 of SEQ ID NO: 38; 
 (l) 28, 254, 309, 407, and 451 of SEQ ID NO: 38; 
 (m) 28, 254, 309, 407, and 480 of SEQ ID NO: 38; or 
 (n) 28, 309, 407, 451, and 480 of SEQ ID NO: 38; 
 wherein the CYP153A-reductase hybrid fusion polypeptide variant catalyzes the conversion of a fatty acid to an omega-hydroxylated fatty acid. 
 
     
     
       4. The CYP153A-reductase hybrid fusion polypeptide variant of  claim 3 , wherein:
 (a) the mutations at amino acid positions 12, 27, 119, 140, 157, 159, 233, and 244 of SEQ ID NO: 38 are Q12W, R27L, K119R, S140N, S157R, V159M, S233L, and A244R, respectively; 
 (b) the mutations at amino acid positions 12, 28, 61, 119, 231, 309, 413, and 480 of SEQ ID NO: 38 are Q12R, Q28M, N61L, K119R, A231V, N309S, Y413R, and I480G, respectively; 
 (c) the mutations at amino acid positions 12, 119, 231, and 480 of SEQ ID NO: 38 are Q12R, K119R, A231V, and I480G, respectively; 
 (d) the mutations at amino acid positions 12, 28, 77, 119, 141, 231 and 407 of SEQ ID NO: 38 are Q12T, Q28M, R77Q, K119R, V141T, A231W and N407G, respectively; 
 (e) the mutations at amino acid positions 12, 28, 61, 141, 231, and 407 of SEQ ID NO: 38 are Q12R, Q28M, N61L, V141T, A231Y, and N407G, respectively; 
 (f) the mutations at amino acid positions 12, 28, 119, 231, and 244 of SEQ ID NO: 38 are Q12W, Q28M, K119R, A231Y, and A244R, respectively; 
 (g) the mutations at amino acid positions 12, 28, 407, and 480 of SEQ ID NO: 38 are Q12W, Q28T, N407G, and I480G, respectively; 
 (h) the mutations at amino acid positions 12, 141, 231, 413, and 481 of SEQ ID NO: 38 are Q12R, V141T, A231Y, Y413R, and G481I, respectively; 
 (i) the mutations at amino acid positions 12, 28, 111, 231, and 407 of SEQ ID NO: 38 are Q12T, Q28M, F111A, A231V, and N407G, respectively; 
 (j) the mutations at amino acid positions 12, 28, 61, 140, and 149 of SEQ ID NO: 38 are Q12T, Q28M, N61L, S140N, and P149R, respectively; 
 (k) the mutations at amino acid positions 12, 28, 77, 119, 159, 231, 254, 407, and 480 are Q12W, Q28T, R77Q, K119R, V159M, A231Y, R254G, N407G, and I480G, respectively; 
 (l) the mutations at amino acid positions 12, 28, 254, 309, 407, and 451 of SEQ ID NO: 38 are Q12W, Q28T, R254G, N309S, N407G, and V451M, respectively; 
 (m) the mutations at amino acid positions 12, 28, 254, 309, 407, and 480 of SEQ ID NO: 38 are Q12W, Q28T, R254G, N309S, N407G, and I480G, respectively; and 
 (n) the mutations at amino acid positions 12, 28, 309, 407, 451, and 480 of SEQ ID NO: 38 are Q12W, Q28T, N309S, N407G, V451M, and I480G, respectively. 
 
     
     
       5. The CYP153A-reductase hybrid fusion polypeptide variant of  claim 1 , wherein expression of the CYP153A-reductase hybrid fusion polypeptide variant in a recombinant host cell results in a higher titer of an omega-hydroxylated fatty acid as compared to the titer of an omega-hydroxylated fatty acid produced by expression of the CYP153A-reductase hybrid fusion polypeptide of SEQ ID NO: 6 or SEQ ID NO: 38 in a corresponding host cell. 
     
     
       6. A recombinant host cell expressing the CYP153A-reductase hybrid fusion polypeptide variant of  claim 1 . 
     
     
       7. The recombinant host cell of  claim 6 , further expressing a thioesterase polypeptide of EC 3.1.2.-, EC 3.1.1.5 or EC 3.1.2.14. 
     
     
       8. The recombinant host cell of  claim 7 , wherein the recombinant host cell produces a omega-hydroxylated fatty acid composition with a titer that is at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, or at least 30% greater than the titer of an omega-hydroxylated fatty acid composition produced by a host cell expressing a corresponding CYP153A-reductase hybrid fusion polypeptide comprising SEQ ID NO: 38 or SEQ ID NO: 6, when cultured in medium containing a carbon source. 
     
     
       9. A method of producing an omega-hydroxylated fatty acid, comprising: (i) culturing the recombinant host cell of  claim 8 , the presence of a carbon source. 
     
     
       10. The CYP153A-reductase hybrid fusion polypeptide variant of  claim 1 , wherein the least one mutation at amino acid position 9 is D9N, or D9K; at amino acid position 10 is D10Y; at amino acid position 11 is I11L; at amino acid position 12 is Q12W, Q12R, or Q12T; at amino acid position 13 is s13K; at amino acid position 14 is R14F; at amino acid position 28 is Q28M, or Q28T; at amino acid position 61 is N61L; at amino acid position 77 is R77Q; at amino acid position 119 is K119R; at amino acid position 159 is V159M; at amino acid position 327 is P327D; at amino acid position 413 is Y413R; at amino acid position 703 is L703G; at amino acid position 745 is P745K, or P745R; at amino acid position 747 is P747N; at amino acid position 749 is E749L, or E749M; at amino acid position 757 is E757A; at amino acid position 770 is T770G; at amino acid position 771 is V771F; or at amino acid position 784 is M784I.